The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that are produced by more than 20 species from the genera Clostridium, Streptococcus, Listeria, Bacillus, and Arcanobacterium. The pore-forming mechanism of these toxins exhibits two hallmark characteristics: an absolute dependence on the presence of membrane cholesterol and the formation of an extraordinarily large pore. Each CDC is produced as a soluble monomeric protein that, with the exception of one member, is secreted by a type II secretion system. Upon encountering a eukaryotic cell, the CDCs undergo a transformation from a soluble monomeric protein to a membrane-embedded supramolecular pore complex. The conversion of the monomers to an oligomeric, membrane-inserted pore complex requires some extraordinary changes in the structure of the monomer.
Although the CDCs are well known as beta-hemolytic proteins, it has become increasingly apparent that bacterial pathogens use these proteins in much more sophisticated ways than as simple hemolysins or general cell-lytic agents. The CDC structure also exhibits a plasticity that has allowed the evolution of unique features for some CDCs, without compromising the fundamental pore-forming mechanism. Some of these features are reflected in CDCs that activate complement, that utilize a nonsterol receptor, that exhibit a pH-sensitive, poreforming mechanism, or that can function as a protein translocation channel.
CDC's are β-sheet-rich, four-domain proteins. A highly conserved tryptophan-rich undecapeptide is present in domain 4, which participates in the binding of some CDCs to cholesterol-rich membranes. In addition, three other short hydrophobic loops (Loops L1, L2 and L3) juxtaposed to the undecapeptide at the tip of domain 4 have been shown to also insert into the membrane surface and anchor the CDC to the membrane in a perpendicular orientation. After membrane binding, the CDC monomers diffuse laterally to initiate formation of the membrane oligomer.
Once the prepore complex reaches a large size, presumably a complete ring structure, it then makes the transition to the pore complex. The transmembrane pore is formed when two α-helical bundles in domain 3 of each monomer within the prepore complex are converted to two extended amphipathic transmembrane β-hairpins (TMHs). Upon the conversion of the prepore to the pore, the height of the prepore structure undergoes a vertical collapse of about 40 Angstroms. The collapse of the prepore structure brings the domain 3 TMHs within striking distance of the membrane surface, at which point they undergo a concerted insertion into the membrane that results in the formation of the large transmembrane β-barrel pore. The CDC pore is large: it is comprised of 35 to 50 monomers and exhibits a diameter of 250 to 300 Angstroms.
During the process of the CDC monomer interaction with the membrane, the undecapeptide and the three other short loops (L1, L2, L3) at the tip of the domain 4 β-sandwich insert into the membrane upon the interaction of the CDC monomers with the membrane surface. These loops do not penetrate deeply into the membrane and apparently do not directly participate in the structure of the transmembrane pore. One function of the loops appears to be to anchor the monomers to the membrane in an upright position. Domain 4 exists in a perpendicular orientation to the membrane and is surrounded by the aqueous milieu, even in the oligomeric state.
Domain 4 of the CDCs mediates membrane recognition, whether it is via cholesterol or another receptor, as in the case of ILY.
The CDCs are also capable of the lysis of a wide variety of nucleated cell types in vitro, and this capacity has in turn has been used by many investigators to permeabilize various eukaryotic cell types with CDCs. Despite the ability of these toxins to perform as general cell-lytic agents in vitro, it has not yet been demonstrated that cell lysis is a primary function of the CDCs during an infection. The contribution of CDCs to infection has been studied for example in Listeria monocytogenes, Streptococcus pyogenes, Streptococcus pneumoniae, Arcanobacterium pyogenes, and Clostridium perfringens. The results of some of these studies suggest that the bacteria use the CDCs in more sophisticated ways than as general cytolytic agents. It also appears that the CDC structure has undergone some unique evolutionary transformations that facilitate the pathogenic mechanism of these bacterial species.
Streptococcus pneumoniae is an important agent of disease in humans, especially among infants, the elderly, persons with chronic illness, and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteremia/septicemia, pneumonia, and meningitis with high morbidity and mortality throughout the world. Even with appropriate antibiotic therapy, pneumococcal infections still result in many deaths. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal disease, the presence of resistant pneumococcal strains has become a major problem in the world today and underscores the need for treating and preventing pneumococcal infection by methods in addition to antimicrobials. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children. New immunogenic pneumococcal vaccines that provide long-term immunity are clearly needed, especially for children aged less than 2 years, because incidence of disease is high and antibody responses to the polysaccharide vaccine antigens are poor in this age group.
Each year in the United States, pneumococcal disease accounts for an estimated 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 7 million cases of otitis media.
Severe pneumococcal infections result from dissemination of bacteria to the bloodstream and the central nervous system. In 1997, data from community-based studies indicated that overall annual incidence of pneumococcal bacteremia in the United States was an estimated 15-30 cases per 100,000 population; the rate was higher for persons aged greater than or equal to 65 years (50-83 cases per 100,000 population) and for children aged less than or equal to 2 years (160 cases per 100,000 population). In adults, 60%-87% of pneumococcal bacteremia was associated with pneumonia; in young children, the primary sites of infection were frequently not identified.
In the United States, the risk for acquiring bacteremia is lower among white persons than among persons in other racial/ethnic groups (i.e., blacks, Alaskan Natives, and American Indians). Black adults have a threefold to fivefold higher overall incidence of bacteremia (49-58 cases per 100,000 population) than whites. Rates of invasive pneumococcal disease are exceptionally high among Alaskan Natives and American Indians. The age-adjusted annual incidence of invasive pneumococcal infection among Alaskan Natives and Alaskan Native children aged less than 2 years was determined by a prospective surveillance study to be 74 cases and 624 cases per 100,000 population, respectively. Rates for meningitis and bacteremic pneumonia are eightfold to tenfold higher for Alaskan Natives of all ages than for other U.S. population groups. The highest incidence rates for any U.S. population have been reported among specific American Indian groups (e.g., Apache). The overall annual incidence for such groups is 156 cases per 100,000 population; the incidence for children aged 1-2 years in these groups is 2,396 cases per 100,000 population.
In the United States, the estimated overall annual incidence of pneumococcal meningitis is one to two cases per 100,000 population. The incidence of pneumococcal meningitis is highest among children aged 6-24 months and persons aged greater than or equal to 65 years. Rates for blacks are twice as high as those for whites and Hispanics. Because the incidence of Haemophilus influenzae type b (Hib) meningitis in children rapidly decreased following the introduction of Hib conjugate vaccines, S. pneumoniae has become the most common cause of bacterial meningitis in the United States (26).
Strains of drug-resistant S. pneumoniae (DRSP) have become increasingly common in the United States and in other parts of the world. In some areas, as many as 35% of pneumococcal isolates have been reported to have intermediate—(minimum inhibitory concentration {MIC}=0.1-1.0 ug/mL) or high-level (MIC greater than or equal to 2 ug/mL) resistance to penicillin. Many penicillin-resistant pneumococci are also resistant to other antimicrobial drugs (e.g., erythromycin, trimethoprim-sulfamethoxazole, and extended-spectrum cephalosporins). High-level penicillin resistance and multidrug resistance often complicate the management of pneumococcal infection and make choosing empiric antimicrobial therapy for suspected cases of meningitis, pneumonia, and otitis media increasingly difficult. Treating patients infected with nonsusceptible organisms may require the use of expensive alternative antimicrobial agents and may result in prolonged hospitalization and increased medical costs. The impact of antimicrobial resistance on mortality is not clearly defined. Emerging antimicrobial resistance further emphasizes the need for preventing pneumococcal infections by vaccination.
The currently available pneumococcal vaccines, manufactured by both Merck and Company, Inc. (Pneumovax 23™) and Lederle Laboratories (Pnu-Immune 23™), include 23 purified capsular polysaccharide antigens of S. pneumoniae (serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F). These vaccines were licensed in the United States in 1983 and replaced an earlier 14-valent formulation that was licensed in 1977. One dose (0.5 mL) of the 23-valent vaccine contains 25 ug of each capsular polysaccharide antigen dissolved in isotonic saline solution with phenol (0.25%) or thimerosal (0.01%) added as preservative and no adjuvant. As of 1997, the 23 capsular types in the vaccine represented at least 85%-90% of the serotypes that cause invasive pneumococcal infections among children and adults in the United States. The six serotypes (6B, 9V, 14, 19A, 19F, and 23F) that most frequently caused invasive drug-resistant pneumococcal infection in the United States as of 1997 are represented in the 23-valent vaccine. As noted below, the desirability of a vaccine solely comprised of capsular polysaccharides is limited.
Pneumolysin in particular is a key component in the pathogenesis of streptococcal pneumonia, which kills over a million humans per year worldwide. The use of pneumolysin as a part of a vaccine for Streptococcus pneumoniae lung infections and otitis media is becoming increasingly important since vaccines based on the capsular polysaccharide are losing effectiveness due to genetic variation and are difficult to generate since there are more than 90 different capsular serotypes of Streptococcus pneumoniae. The immunity to one capsular type does not protect against another capsular type. The currently available pneumococcal vaccine discussed above, which comprises 23 capsular polysaccharides from the strains that most frequently cause disease, has significant shortcomings related primarily to the poor immunogenicity of some capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. Hence, the focus for vaccine development has shifted away from the bacterial capsule to various pneumococcal proteins, pneumolysin being considered as a necessary component for all subunit vaccines. Currently, a single mutant of pneumolysin has been used for vaccine development. This pneumolysin mutant (referred to as “Pd-B”) contains a single mutation at position 433 (wherein the native tryptophan residue has been changed to a phenylalanine). This mutation in pneumolysin is in the conserved undecapeptide of Domain 4, the structure within the cholesterol-dependent cytolysins (CDCs), which has long been thought to mediate binding to mammalian membranes. Other mutants of pneumolysin are described in U.S. Pat. No. 6,716,432, for example.
While the pneumolysin Pd-B mutant is conventionally used for vaccine development, this protein is still able to undergo a variety of structural transitions that occur after binding to the membrane of mammalian cells. These changes dramatically alter its structure and may decrease its ability to stimulate an effective neutralizing immune response in a patient, primarily because the structure of pneumolysin that the patient's immune system may “see” will be that of the terminal cell-bound oligomeric complex instead of the initial structure of the soluble monomeric pneumolysin. More importantly, the current genetically toxoided pneumolysin is still hampered by an unacceptable level of toxicity. The basis for this toxicity is not yet clear, but likely results from the fact that this toxoid can still bind to and oligomerize on mammalian cells.
Therefore, mutants of cholesterol-dependent cytolysins, such as pneumolysin, which are both non-toxic and which do not bind to cell membranes and which are non-hemolytic yet which stimulate an immune response against corresponding disease organisms would be of great benefit.